The failure to establish proof-of-concept in several clinical trials for the prevention of sexual HIV transmission using microbicides, and the highly complex environment for non-vaccine biomedical prevention (nBP) development and product use underscores the pressing need for an improved understanding of the fundamental processes at work. A theoretical conceptualization of host, drug, and virus interactions can inform the construction of a mechanistic pharmacokinetic-pharmacodynamic (PK-PD) model to support rational nBP development. For topical vaginal delivery, a PK-PD model must encompass the anatomic ?compartments? where the drug and virus distribute. An understanding of drug concentrations over time in these compartments is critical in the development of a useful PK model where knowledge of dosing regimen and concentration in one compartment allows estimation of concentration in another compartment, without additional sampling. The Program?s long-term goal is to develop intravaginal ring (IVR) formulations of multiple antiretroviral (ARV) agents for prevention of sexual HIV infection, emphasizing the needs of women in the developing world. The objective of this application is to quantitatively explore the PK domain in animals and humans receiving IVRs formulated with combinations of up to three ARV drugs by sampling of up to 6 different compartments within the body. In Aim 1, assays will be developed and validated for all studied ARV drugs in the 6 target compartments (fluid and CD4+ cell components, where relevant, within the cervicovaginal secretions, tissues, and blood). The PK for optimized IVR formulations will be determined in mice, sheep, macaques, and humans in a (pre-Phase I) Exploratory Clinical Trial (Project 4). In Aim 2, the spatiotemporal drug concentration data gathered will be evaluated in light of PD outcomes, both efficacy (Project 3) and toxicity (Project 2): ex vivo, in a novel 3D vaginal epithelial cell (VEC) culture model; in vivo, in sheep (toxicity only), humanized mice, and macaques; and humans (Project 4) for toxicity and surrogate efficacy. The results will be used to develop exploratory PK-PD models to describe the drug exposure-response relationships for each of the candidate ARV IVR formulations tested. In Aim 3, the role of membrane transporters and metabolizing enzymes in the vaginal epithelium on drug disposition and potential drug-drug interactions will be investigated ex vivo using our VEC culture system and samples collected as part of the above in vivo studies.